补骨脂素联合长波紫外线A诱导HL-60细胞凋亡作用的研究

本研究探讨中药补骨脂素（psoralen，PSO）加长波紫外线A（ultraviolet A，UVA）（PUVA）诱导人白血病细胞HL-60凋亡的作用及其可能的作用机制。采用MTT法观察PUVA对HL-60细胞增殖的影响；采用电子显微镜技术观察细胞超微结构改变；流式细胞术（FCM）检测细胞凋亡率、线粒体跨膜电位水平以及细胞Fas、FasL蛋白的表达；荧光定量PCR技术检测细胞Fas、FasL mRNA的表达；免疫细胞化学法（immunocytochemistry，ICC）检测caspase 8、caspase3蛋白的表达。结果表明，PUVA可抑制HL-60细胞的增殖，使凋亡率增加，作用呈时间、浓度依赖性；UVA照射时间15分钟和PSO浓度为80μg／ml时，HL-60细胞增殖的抑制率、凋亡率达峰值；PUVA作用后HL-60细胞超微结构出现明显的凋亡形态学改变，细胞线粒体跨膜电位水平下降；PUVA作用4小时Fas mRNA的表达升高，FasL mRNA的表达下降；PUVA作用24小时Fas、FasL在蛋白水平的表达亦呈现相同规律；PUVA可使HL-60细胞caspase 8、caspase 3蛋白的表达增强，在作用后8小时强度达峰值。结论：PUVA能够抑制HL-60细胞的增殖，并诱导其凋亡，可能的机制是PUVA作用于Fas／FasL系统，使Fas基因表达升高、FasL基因表达下降，激活下游caspase 8、caspase 3的表达，线粒体膜电位水平降低亦可能参与了这个过程。

Effect of Psoralen Plus Longwave UVA Inducing HL-60 Cells Apoptosis

The aim of this study was to investigate the effects of the traditional Chinese medicine psoralen （PSO） plus long wave ultraviolet A（PUVA） on apoptosis in HL-60 leukemia cells and its mechanism. The effect of PUVA on HL-60 cell growth was assayed by MTT method and the changes of ultrastructure of cells were observed by electron microscopy. The apoptosis ratios, changes of mitochondrial membrane potential, expression of Fas and FasL protein and Fas and FasL mRNA were detected by FCM and fluorescent quantitation RT-PCR respectively. The expression of Caspase 8 and Caspase 3 protien were detected by immunocytochemistry （ICC）. The results showed that the growth of HL-60 cells were inhibited by PUVA in time-and concentration-dependant manner through inducing cell apoptosis. When the irradiation time of long wave ultraviolet A lasted 15 minutes and the concentration of PSO was 80 μg/ml, the inhibition of HL-60 cell proliferation and apoptosis ratios reached the peak. There were obvious apoptotic ultrastructure changes and decrease of mitochondrial membrane potential in HL-60 cells after treatment with PUVA. The expression of Fas mRAN increased and expression of FasL mRNA decreased after treating with PUVA for 4 hours, and the same results of Fas, FasL expression on protein level were obtained also after treating with PUVA for 24 hours. The expression of caspase 8 and caspase 3 protien enhanced and reached the peak after treating with PUVA for 8 hours. It is concluded that the PUVA can inhibit the growth of HL-60 cells and induce apoptosis of these cells. The possible mechanism is supposed to be up-regulating Fas, down-regulating FasL levels and then activating the levels of caspase 8 and caspase 3. The decreasing of mitochondrial membrane potential may be involved in this process probably.